Project Summary Membrane fusion, mediated by HIV-1 envelope glycoprotein [Env; trimeric (gp160)3 cleaved to (gp120/gp41)3], is the first critical step for the virus to enter host cells and establish infection. A mature Env spike contains three copies each of noncovalently-associated receptor-binding subunit gp120 and fusion subunit gp41. A general picture of viral membrane fusion has emerged from extensive biochemical and structural studies. Sequential binding of gp120 to the primary receptor CD4 and a coreceptor leads to large, irreversible structural rearrangements in gp41, which drive the membrane fusion process. Despite tremendous progress in our understanding of the structure of HIV-1 Env over the last two decades, largely based on studies of its soluble fragments, we still lack an atomic picture of the full-length Env in a membrane environment. In a series of recent studies, we have determined the structures of the transmembrane domain, membrane proximal external region, as well as a portion of the cytoplasmic tail of HIV-1 Env in bicelles that mimic lipid bilayers by NMR. Unexpectedly, we find that these regions all form well-ordered trimeric clusters in the presence of a lipid bilayer and that disruption of any of them reduces membrane fusion efficiency and alters the antigenic structure of the entire Env, suggesting that they play critical structural and functional roles. These new findings provide a strong scientific premise to determine the structure of the full-length HIV-1 Env reconstituted in lipid nanodiscs by cryo-electron microscopy (cryoEM). We hypothesize that the transmembrane and membrane- proximal regions of HIV-1 Env all adopt defined oligomeric structures that are critical for the stability, function and antigenicity of the full-length protein in membrane. We will capitalize on the recent advances in cryoEM and nanodisc technology and plan to determine structures of the full-length Env proteins, reconstituted in lipid bilayers, both alone or in complex with the matrix protein. Our goal is to visualize novel structural features of the intact Env proteins in the context of membrane, to gain a full understanding of their structure-function and to facilitate Env-based immunogen design for vaccine development. We will purse the following specific aims: 1) we will determine the structure of a full-length HIV-1 Env in the context of membrane, 2) we will determine the structural basis for antigenic differences of the intact Env in membrane among HIV-1 isolates with different antibody sensitivity, and 3) we will determine the structure of a full-length HIV-1 Env in complex with matrix protein.